Three-photon light-sheet fluorescence microscopy

Escobet-Montalbán, Adrià; Liu, Pengfei; Nylk, Jonathan; Gasparoli, Federico M.; Yang, Zhengyi; Dholakia, Kishan

doi:10.1101/323790

Cited by 10 publications

(13 citation statements)

References 22 publications

(10 reference statements)

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“…Since the first demonstrations of 3PM for in vivo brain imaging (Horton et al, 2013; Ouzounov et al, 2017), a number of research groups have successfully adopted and developed the technology (Chen et al, 2018; Escobet-Montalbán et al, 2018; Odríguez, 2018; Perillo et al, 2017; Rowlands et al, 2017; Takasaki et al, 2019b; Weisenburger et al, 2019; Yildirim et al, 2019), propelled by the commercially available lasers and microscopes. To facilitate 3PM applications, here we provide a quantitative characterization of three-photon deep-brain calcium imaging, with a side-by-side comparison to 2PM with 920 nm excitation.…”

Section: Introductionmentioning

confidence: 99%

Quantitative Analysis of 1300-nm Three-photon Calcium Imaging in the Mouse Brain

Wang

Ouzounov

et al. 2019

Preprint

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1300-nm three-photon calcium imaging has emerged as a useful technique to allow calcium imaging in deep brain regions. Application to large-scale neural activity imaging entails a careful balance between recording fidelity and tissue heating. We calculated and experimentally verified the excitation pulse energy to achieve the minimum photon count required for the detection of calcium transients in GCaMP6s-expressing neurons for 920-nm two-photon and 1320-nm three-photon excitation, respectively. Brain tissue heating by continuous three-photon imaging was simulated with Monte Carlo method and experimentally validated with immunohistochemistry. We observed increased immunoreactivity with 150 mW excitation power at 1.0- and 1.2-mm imaging depths. Based on the data, we explained how three-photon excitation achieves better calcium imaging fidelity than two-photon excitation in the deep brain and quantified the imaging depth where three-photon microscopy should be applied. Our analysis presents a translatable model for the optimization of three-photon calcium imaging based on experimentally tractable parameters.

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Section: Introductionmentioning

confidence: 99%

Quantitative Analysis of 1300-nm Three-photon Calcium Imaging in the Mouse Brain

Wang

Ouzounov

et al. 2019

Preprint

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“…It has also been shown that by relying on such higher order nonlinear process, fluorescence is confined to a smaller volume, reducing out-of-focus light. 17,22 Novel laser sources have recently been developed to generate high energy pulses to optimize the absorption of three photons. Such ultrashort Compressive sensing is demonstrated a posteriori, by using 5% and 15% of the recorded Hadamard patterns and minimizing to the l1 norm.…”

Section: Three-photon Trafixmentioning

confidence: 99%

“…However, conventional Ti:Sapphire lasers can still be used to excite fluorophores at the high frequency end of the visible spectrum. 22 To demonstrate that TRAFIX can potentially be used in 3P excitation mode, we imaged 2.1 um blue fluorescent microspheres. To obtain a high power per unit area and achieve enough photon density to generate 3P excitation, the FOV was reduced to only 20 µm × 20 µm.…”

Section: Three-photon Trafixmentioning

confidence: 99%

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Wide-field multiphoton imaging with TRAFIX

Escobet-Montalbán

Wijesinghe

Chen

et al. 2019

Multiphoton Microscopy in the Biomedical Sciences XIX

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Optical approaches have broadened their impact in recent years with innovations in both wide-field and superresolution imaging, which now underpin biological and medical sciences. Whilst these advances have been remarkable, to date, the ongoing challenge in optical imaging is to penetrate deeper. TRAFIX is an innovative approach that combines temporal focusing illumination with single-pixel detection to obtain wide-field multiphoton images of fluorescent microscopic samples deep through scattering media without correction. It has been shown that it can image through biological samples such as rat brain or human colon tissue up to a depth of seven scattering mean-free-path lengths. Comparisons of TRAFIX with standard point-scanning two-photon microscopy show that the former can yield a five-fold higher signal-to-background ratio while significantly reducing photobleaching of the specimen. Here, we show the first preliminary demonstration of TRAFIX with three-photon excitation imaging dielectric beads. We discuss the advantages of the TRAFIX approach combined with compressive sensing for biomedicine.

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“…The NIR excitation allows for improved penetration into biological samples and minimises scattering compared to visible excitation 25 . Twophoton light sheet fluorescence microscopy (2P-LSFM) has been used for live imaging of zebrafish development 7 , and more recently three-photon light sheet fluorescence microscopy (3P-LSFM) has been used in conjunction with Bessel beam illumination for 3D imaging with high-contrast and low photodamage in highly scattering cell spheroids over a large FOV 26 . While with one-photon (1P) excitation the out-of-focus side-lobes in Airy and Bessel beams cause a reduction of image contrast, with 2P excitation, due to the quadratic dependence on incident intensity, this issue gets largely resolved.…”

Section: Introductionmentioning

confidence: 99%

Label-free and Multimodal Second Harmonic Generation Light Sheet Microscopy

Hanrahan

Lane

Johnson

et al. 2020

Preprint

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Light sheet microscopy (LSM) has emerged as one of most profound three dimensional (3D) imaging tools in the life sciences over the last decade. However, LSM is currently performed with fluorescence detection on one- or multi-photon excitation. Label-free LSM imaging approaches have been rather limited. Second Harmonic Generation (SHG) imaging is a label-free technique that has enabled detailed investigation of collagenous structures, including its distribution and remodelling in cancers and respiratory tissue, and how these link to disease. SHG is generally regarded as having only forward- and back-scattering components, apparently precluding the orthogonal detection geometry used in Light Sheet Microscopy. In this work we demonstrate SHG imaging on a light sheet microscope (SHG-LSM) using a rotated Airy beam configuration that demonstrates a powerful new approach to direct (without any further processing or deconvolution) 3D imaging of harmonophores such as collagen fibres in biological samples. We provide unambiguous identification of SHG signals on the LSM through its wavelength and polarisation sensitivity. In a multimodal LSM setup we demonstrate that SHG and two-photon signals can be acquired on multiple types of different biological samples. We further show that SHG-LSM is sensitive to changes in collagen synthesis within lung fibroblast 3D cell cultures. This work expands on the existing optical methods available for use with light sheet microscopy, adding a further label-free imaging technique which can be combined with other detection modalities to realise a powerful multi-modal microscope for 3D bioimaging.

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Three-photon light-sheet fluorescence microscopy

Cited by 10 publications

References 22 publications

Quantitative Analysis of 1300-nm Three-photon Calcium Imaging in the Mouse Brain

Quantitative Analysis of 1300-nm Three-photon Calcium Imaging in the Mouse Brain

Wide-field multiphoton imaging with TRAFIX

Label-free and Multimodal Second Harmonic Generation Light Sheet Microscopy

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